Natural killer T (NKT) cells represent a subset of T lymphocytes expressing both T-cell receptor and NK-cell receptor, and play a role in bridging innate immunity to adaptive immunity. Kronenberg M and Gapin L, Nat Rev Immunol 2: 557-568 (2002). Upon activation, NKT cells can have a pronounced impact on early and delayed immunity to various pathogens, including L. monocytogenes, M. tuberculosis and Leishmania major. Kronenberg (2002); Behar S M and Porcelli S A, Curr Top Microbiol Immunol 314: 215-250 (2007); Emoto M et al., Eur J Immunol 29: 650-659 (1999); Ishikawa H et al., Int Immunol 12: 1267-1274 (2000); and Ranson T et al., J Immunol 175: 1137-1144 (2005). NKT cell activation has been reported to lead to enhanced CD4 and CD8 T cell responses, and to induce dendritic cell (DC) maturation. Nishimura T et al., Int Immunol 12: 987-994 (2000) and Silk J D et al., J Clin Invest 114: 1800-1811 (2004).
Unlike conventional T cells that recognize MHC-bound peptides, NKT cells are specific for lipid antigens presented by the MHC class I-like protein CD1d. Several glycolipid antigens, including self-derived and bacterial-derived glycolipids, which can be presented by CD1d to activate NKT cells, have been identified to date. Tsuji M Cell Mol Life Sci 63: 1889-1898 (2006). NKT cells that have T-cell receptors with invariant Vα14-Jα18 rearrangements (iNKT cells) possess reactivity to a glycosphingolipid, α-galactosylceramide (αGalCer), when presented by CD1d. Kronenberg M and Gapin L, Nat Rev Immunol 2: 557-568 (2002); Kronenberg M, Annu Rev Immunol 23: 877-900 (2005). Recent studies have shown that vaccines against Plasmodia, Leishmania donovanii, Listeria monocytogenes and HIV could be improved by activating iNKT cells through co-administration of αGalCer as an adjuvant. Gonzalez-Aseguinolaza G et al., J Exp Med 195: 617-624 (2002); Dondji B et al., European Journal of Immunology 38: 706-719 (2008); Huang Y X et al., Vaccine 26: 1807-1816 (2008); and Enomoto N et al., FEMS Immunol Med Microbiol 51: 350-362 (2007).
As a therapeutic, αGalCer has been shown to reduce malarial parasite load in mice and prolong the survival of M. tuberculosis infected mice. Gonzalez-Aseguinolaza G et al., Proc Natl Acad Sci USA 97: 8461-8466 (2000); Chackerian A et al., Infection and Immunity 70: 6302-6309 (2002). A single injection of αGalCer in mice induces secretion of IFNγ, IL-12 and IL-4 in serum. Fujii S et al., Immunol Rev 220: 183-198 (2007). Stimulation of CD1d-restricted iNKT cells by αGalCer also leads to a rapid cascade of activation of immune and inflammatory cells including NK cells, dendritic cells, B cells, and conventional T cells. Nishimura T et al., Int Immunol 12: 987-994 (2000); Kitamura H et al., J Exp Med 189: 1121-1128 (1999); Fujii S et al., J Exp Med 198: 267-279 (2003). iNKT cells produce large amounts of IL-4 and IFNγ and the production requires direct contact between iNKT cells and DCs through CD40-CD40 ligand interactions. Nishimura T et al., Int Immunol 12: 987-994 (2000). IFNγ produced by iNKT cells has been shown to have a critical role in the antimetastatic effect of αGalCer in murine tumor models. Hayakawa Y et al., Eur J Immunol 31: 1720-1727 (2001); Smyth M J et al., Blood 99: 1259-1266 (2002). Thus, activation of iNKT cells may play a role in modulating adaptive immune responses by influencing the early cytokine environment, thereby enhancing host resistance to infectious and inflammatory diseases.
Soluble CD1d proteins loaded with glycolipids such as α-galactosylceramides are being developed as immunotherapeutic agents. However, their utility is limited by the instability of the noncovalent interaction between the glycolipid and the protein, which allows them to rapidly dissociate or be displaced by natural competitive inhibitors and lose activity in vivo. Webb T J et al. Cancer Res 72: 3744-3752 (2012). There remains a need to develop a method for covalently linking the glycolipid to the CD1d protein to create stable and long-lived complexes that maintain potent NKT-cell activating properties.